This invention relates to the use of a heterocyclic diazo dye for the cytological preparation of a biopsy specimen derived from blood, bone marrow, lymph nodes, and other specimens of hematopoietic origin. More specifically, this invention relates to the use of a basic cationic diazo dye known as "Basic Blue 93" for staining dried or fixed specimens of human blood and bone marrow cells. The diazo dye reacts with the lysosomes (primary granules) in the cells of the neutrophilic granulocytic series providing means to identify and differentiate between the myeloblasts and the lymphoblasts prior to the treatment of leukemic patients.
The use of cationic water-soluble heterocyclic diazo dyes of this invention is an advance over the prior art (Romanowsky and Malachowski) where mixtures of dyes were used for staining biopsy specimens. The stained cells of hematopoietic origin obtained in accordance with this invention provide excellent color stability and are exceptionally clear with respect to cellular detail and brilliance of the cell structure.
The diagnosis of hematological disorders has been accomplished, in part, by the enumeration and identification of formed elements of the peripheral blood and bone marrow. The basis of hematological diagnosis has been microscopic examination of a panoptically stained specimen of blood or marrow which provides sufficient information to make a diagnosis. In addition to the developments in panoptic light microscopy, cytochemical stains were developed to identify cell types more precisely than was possible by using panoptic stains. Cytochemistry is biochemistry on a microscopic and submicroscopic level applied to cells and tissues. The cytochemical stains identify enzymes, substrates, and organelles. Cytochemical stains can be selective for one cell type compared to another and, as such, these stains have been used as diagnostic tools, especially in making distinctions between the various cytologic types of acute leukemias and preleukemic disorders.
With the development of new synthetic organic dyestuffs, it was found that, by adding these dyes to freshly obtained specimens of blood, some cells stained with one or more dyes, where others did not. Interest in supravital stains is based on the ability of some of these dyes to stain a reticulated network in young erythrocytes. The demonstration of reticulocytes by the supervital technique is still a standard laboratory test in hematological diagnosis.
Following the developments in supravital staining of blood, Ehrlich appreciated the need for more stable, competent preparations of blood that could be examined under the microscope. Ehrlich devised stains comprising Orange G, acid fuschin, and methyl green. By the use of this mixture of dyes, Ehrlich identified and named most of the blood leukocytes based on differential staining as we know them today. With the recognition that some of the available dyes caused differential coloration of blood cells, others modified Ehrlich's stain, including, for example, the Romanowsky method, followed by Wright, Jenner, May, and Grunwald and Giemsa's method. All of these modifications included a mixture comprising several basic cationic dyes.
It was discovered early in the history of morphologic and cytochemical diagnosis of blood disorders that the examination of only panoptically stained specimens of blood or bone marrow was not sufficient to make a diagnosis. By using cytochemical stains, it is possible to identify the presence or absence of substance in one cell type in comparison to another, or the increase or decrease in the quantity of a substance in these cell types. These differences are important diagnostic tools when they reflect differences in one cell type compared to another, and in normal cells compared to pathologic blood cells. It is now appreciated that quantitative differences in the intensity of cytochemical stains are important diagnostically. Moreover, the differences in the configuration and spatial distribution of the reaction product can have diagnostic value.
In erythroblasts, for example, of a patient with erythroleukemia, a PAS (periodic acid-Schiff) stain may reveal large chunklike aggregates of glycogen. In patients with acute lymphoblastic leukemia, the glycogen may appear as smaller punctate aggregates. In patients with chronic erythremic myelosis, the pattern of the staining may be diffused and punctate in the same cell, or either diffused or punctate in different cells. The differences in the configuration and spatial distribution of glycogen in different pathologic blood cells may be relevant to the metabolic abnormalities that are unique to each type of cell.
Early in the study of blood cell identification, it became apparent that the differentiation and identification of the early immature or primitive precursor cells in the blood and bone marrow created difficulties when using the Romanowsky type of stains. To circumvent this problem, the cytochemical stains were developed which, when applied to blood and bone marrow cells, identified the mature cells and their primitive or immature precursors on the basis of a characteristic property, such as a unique enzyme or cellular metabolic, rather than on the physical features, such as size, shape, and color. The earliest cytochemical stains to be applied to the blood and bone marrow cell identification were the peroxidase stains. It has become apparent that some cells contain peroxidase activities (granulocytes) while other cells (lymphocytes) do not. Not long afterwards, the peroxidase stain was used in the study of immature or permanent cells in acute leukemia. Thus, for the first time it became possible to distinguish immature or primitive granulocytic cells, i.e., myeloblasts, from immature lymphoid cells or lymphoblasts. This distinction could be achieved on the basis of the detection of myeloperoxidase activity in myeloblasts, but not in lymphoblasts.
It is a practice in cytochemical staining of myeloperoxidase to add a color-forming compound, and exogenous chromogen, such as benzidene or o-tolidine coupled with an oxidizing agent, such as hydrogen peroxide. Benzidene, however, is a carcinogen, and therefore a potential hazard to the user. The reaction product obtained is, unfortunately, unstable, and fades over a period of time. Moreover, some of the leukemic myeloblasts presently being identified as myeloblasts with monocolonal antibodies do not contain a demonstrable peroxidase activity. Over the past several decades, other tests have been devised as alternatives to the myeloperoxidase reaction because of these limitations.
One of the alternative tests was the use of a Sudan Black stain. The Sudan Black stain is known as CI 26150. When applied to fixed preparations of blood and bone marrow cells, Sudan Black B stains lipids in the granules of granulocytic cells at all stages of their development. The stain is weakest when used in conjunction with immature granulocytic cells, like myeloblasts and promyelocytes. It is strongest in mature granulocytic cells, such as neutrophils, eosinophils, and basophils. Although useful in the laboratory for distinctive identification between leukemic myeloblasts and leukemic lymphoblasts, Sudan Black B stains show poor localization in granular structures. Moreover, there is considerable non-specific background precipitation of the dye in many of the contemporary methods where it is used.
Along with the development of Sudan Black B as an alternative to the traditional myeloperoxidase reaction, a specific esterase enzyme was identified as unique to the cells of the granulocytic series. By using naphthol-ASD-chloroacetate as the substrate and a sensitized dye such as Blue BBN as the indicator, the specific esterase stain demonstrates granulocytic properties in immature cells like the myeloblasts in acute myeloblastic leukemias, or in granulocytic sarcoma (chloroma). This stain and methods of using the stain for this purpose have several shortcomings, which include the need for an exogenous substrate, with a sensitized unstable dye coupler, imprecise localization of the reaction product in granules, need for a nuclear counterstain, prolonged incubation period to perform the test, and considerable background precipitation of the coupler, making it difficult to distinguish between artifact and the reaction product.
As a result of these shortcomings of the specific esterase reaction, other stains have been developed as an alternative. Used as a direct stain applied to a fixed preparation of blood and bone marrow cells, these stains were simpler for application than the traditional stains, and have produced comparable results. Complexities associated with the specific esterase reaction tests led to the discovery that chlorozol Black E (direct Black 38, CI 30235) wherein both the primary granules or lysosomes, and secondary granules or specific granules have been distinguished and demonstrated in neutrophilic granulocytic cells on the basis of identifiable differences. Using Saturn Blue, CI 42045, also known as "Acid Blue 1," similar differences in the primary and secondary granules have been noted. In the use of Niagara Sky Blue 6B, CI 24410, known also as "Direct Blue 1," identifiable differential coloration of primary and secondary granules provided an advance in the art. With an acid dye, known as "Sulfonaphthyl Red," an acid dye not identified by the Color Index number, both primary and secondary granules are stained red. As presently known, there is no dyestuff reported which will selectively and preferentially stain only the primary granules or lysosomes in a dried and/or fixed cell of the neutrophilic granulocytic series, including the myeloblast, promyelocytes, myelocytes, bands, and neutrophils. None of the dyes known heretofore have been sufficiently selective, since they stained both the primary and secondary granules, producing in some instances a color difference.
Accordingly, it is an object of this invention to provide a water-soluble cationic diazo dye useful in staining unfixed and/or fixed cells of the neutrophilic granulocytic series.
It is another object of this invention to provide cells of a hematopoietic origin fixed and subsequently stained with a water-soluble cationic diazo dye to obtain stained cells having individual color characteristics which permit the differentiation, identification, and enumeration thereof by use of an optical instrument.
It is still a further object of this invention to provide a plurality of cells of hematopoietic origin stained with effective amounts of water-soluble cationic diazo dyes.
These and other objects of the invention will become apparent from a further and more detailed description of the invention.